Method for measuring concentration of analyte

ABSTRACT

A biosensor system can comprise a sensor chip and a measurement device. The sensor chip comprises a capillary and electrodes disposed within the capillary. The height of the capillary is set to be less than the maximum value of the sum of the diffusion distance of an electron-transfer mediator and the diffusion distance of an analyte at the upper limit of the measurement guaranteed temperature of the biosensor system. The measurement device applies an open circuit voltage, a voltage that is lower than during concentration measurement, or the like to the electrodes of the sensor chip.

TECHNICAL FIELD

The present invention relates to a biosensor system and to a method formeasuring the concentration of an analyte.

BACKGROUND ART

A portable biosensor system comprising a measurement device with acomputer, and a sensor chip that can be installed in this measurementdevice, has been used in the past to measure the concentration of ananalyte in a blood sample, such as the blood glucose concentration(glucose value). The concentration of the analyte is calculated on thebasis of the amount of oxidized product and reduced product produced byan enzyme cycling reaction via a redox enzyme in which the analyteserves as the substrate. The speed of the enzyme cycling reactiondepends on the temperature of the environment in which the reactiontakes place (the reaction temperature). Accordingly, a biosensor systemhas been proposed that comprises a function of correcting theconcentration of an analyte on the basis of the reaction temperature.The reaction temperature is measured, for example, by a temperaturesensor disposed in the measurement device (Patent Literature 1).

CITATION LIST Patent Literature

Patent Literature 1: Japanese Laid-Open Patent Application 2003-156469

SUMMARY Technical Problem

With the biosensor system of Patent Literature 1, the internaltemperature of the measurement device is measured with a temperaturesensor. Thus, the measured temperature does not accurately reflect thetemperature of the blood sample. Accordingly, error may occur in themeasurement of the analyte concentration.

It is an object of the present invention to provide a biosensor systemand a concentration measurement method with which error is less likelyto be caused by temperature changes.

Solution to Problem

The biosensor system pertaining to a first aspect of the presentinvention is a biosensor system with which the concentration of ananalyte in a liquid sample is measured using a redox enzyme or anelectron-transfer mediator, said biosensor system comprising a sensorchip comprising a capillary into which a liquid sample is introduced,whose height is less than the maximum value of the sum of the diffusiondistance of the electron-transfer mediator and the diffusion distance ofthe analyte at the upper limit of the measurement guaranteed temperatureof the biosensor system, a plurality of electrodes disposed within thecapillary, and a reagent layer that is disposed within the capillary andincludes the electron-transfer mediator; a first voltage applicator thatapplies a first voltage to the electrodes; a concentration measurementsection that measures the concentration of the analyte on the basis ofthe value of the current flowing through the liquid sample during thefirst voltage application; and a second voltage applicator that appliesa second voltage to the electrodes prior to the application of the firstvoltage, so that the effect of the temperature of the liquid sample onthe measurement results of the concentration measurement section will bediminished.

The measurement method pertaining to a second aspect of the presentinvention is a method for measuring the concentration of an analyte in aliquid sample using a redox enzyme or an electron-transfer mediator,which is executed by a biosensor system having a sensor chip comprisinga capillary into which a liquid sample is introduced, whose height isless than the maximum value of the sum of the diffusion distance of theelectron-transfer mediator and the diffusion distance of the analyte atthe upper limit of the measurement guaranteed temperature of thebiosensor system, a plurality of electrodes disposed within thecapillary, and a reagent layer that is disposed within the capillary andincludes the electron-transfer mediator, said measurement methodcomprising a first voltage application step of applying a first voltageto the electrodes, a current detection step of detecting the value ofcurrent flowing through the liquid sample during the application of thefirst voltage, a concentration measurement step of measuring theconcentration of the analyte on the basis of the current value, and asecond voltage application step of applying a second voltage to theelectrodes prior to the detection of the current value, so that thetemperature of the liquid sample will have less effect on themeasurement results of the concentration measurement section.

Advantageous Effects

With the biosensor system and measurement method pertaining to thepresent invention, the distance that an analyte in a liquid sample canmove by diffusion is limited by a capillary. Furthermore, a secondvoltage is applied to electrodes before a first voltage is applied,which reduces variance in the measurement result caused by temperature.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is an perspective view of the configuration of a biosensor systempertaining to an embodiment of the present invention;

FIG. 2 is an exploded perspective view of a sensor chip included in thebiosensor system in FIG. 1;

FIG. 3 is a plan view of the sensor chip in FIG. 2;

FIG. 4 is a schematic diagram of the diffusion distance of an analyteand a mediator;

FIG. 5A is a diagram illustrating the height of a capillary in thesensor chip pertaining to an embodiment;

FIG. 5B is a diagram illustrating the height of a capillary in thesensor chip pertaining to another embodiment;

FIG. 6 is a diagram of the internal configuration of a measurementdevice 101 in the biosensor system in FIG. 1;

FIG. 7 is a flowchart showing an example of the flow in a method formeasuring the concentration of a blood sample with the biosensor systemin FIG. 1;

FIG. 8 is a flowchart showing another example of the flow in a methodfor measuring the concentration of a blood sample;

FIG. 9 is a flowchart showing yet another example of the flow in amethod for measuring the concentration of a blood sample;

FIG. 10A is a graph of an example of the pattern of voltage applicationto a sensor chip;

FIG. 10B is a graph of another example of the pattern of voltageapplication to a sensor chip;

FIG. 10C is a graph of yet another example of the pattern of voltageapplication to a sensor chip;

FIG. 10D is a graph of yet another example of the pattern of voltageapplication to a sensor chip;

FIG. 11A is a graph of the response current value when the glucoseconcentration of the sample is 100 mg/dL (milligrams per deciliter),neither the application of open circuit voltage nor the application oflow voltage is executed, the applied voltage is 250 mV, and the heightof the capillary is 150 μm;

FIG. 11B is a graph of the response current value under the sameconditions as in FIG. 11A, except that the height of the capillary is100 μm;

FIG. 11C is a graph of the response current value under the sameconditions as in FIG. 11A, except that the height of the capillary is 59μm;

FIG. 11D is a graph of the response current value under the sameconditions as in FIG. 11A, except that the height of the capillary is 33μm;

FIG. 12A is a graph of the response current value when 8 seconds haveelapsed in FIGS. 11A to 11D;

FIG. 12B is a graph of the variance in the response current value underthe various temperature conditions in FIG. 12A, using the responsecurrent value at 21° C. as a reference;

FIG. 13A is a graph of the response current value when the glucoseconcentration of the sample is 400 mg/dL, neither the application ofopen circuit voltage nor the application of low voltage is executed, theapplied voltage is 250 mV, and the height of the capillary is 150 μm;

FIG. 13B is a graph of the response current value under the sameconditions as in FIG. 13A, except that the height of the capillary is100 μm;

FIG. 13C is a graph of the response current value under the sameconditions as in FIG. 13A, except that the height of the capillary is 59μm;

FIG. 13D is a graph of the response current value under the sameconditions as in FIG. 13A, except that the height of the capillary is 33μm;

FIG. 14A is a graph of the response current value when 8 seconds haveelapsed in FIGS. 13A to 13D;

FIG. 14B is a graph of the variance in the response current value underthe various temperature conditions in FIG. 14A, using the responsecurrent value at 21° C. as a reference;

FIG. 15A is a graph of the amount of charge obtained by adding up themeasurement results for response current value in FIG. 11A every 0.1second;

FIG. 15B is a graph of the amount of charge obtained by adding up themeasurement results for response current value in FIG. 11B every 0.1second;

FIG. 15C is a graph of the amount of charge obtained by adding up themeasurement results for response current value in FIG. 11C every 0.1second;

FIG. 15D is a graph of the amount of charge obtained by adding up themeasurement results for response current value in FIG. 11D every 0.1second;

FIG. 16A is a graph of the amount of charge when 8 seconds have elapsedin FIGS. 15A to 15D;

FIG. 16B is a graph of the variance in the amount of charge under thevarious temperature conditions in FIG. 16A, using the amount of chargeat 21° C. as a reference;

FIG. 17A is a graph of the amount of charge obtained by adding up themeasurement results for response current value in FIG. 13A every 0.1second;

FIG. 17B is a graph of the amount of charge obtained by adding up themeasurement results for response current value in FIG. 13B every 0.1second;

FIG. 17C is a graph of the amount of charge obtained by adding up themeasurement results for response current value in FIG. 13C every 0.1second;

FIG. 17D is a graph of the amount of charge obtained by adding up themeasurement results for response current value in FIG. 13D every 0.1second;

FIG. 18A is a graph of the amount of charge when 8 seconds have elapsedin FIGS. 17A to 17D;

FIG. 18B is a graph of the variance in the amount of charge under thevarious temperature conditions in FIG. 18A, using the amount of chargeat 21° C. as a reference;

FIG. 19A is a graph of the response current value when the glucoseconcentration of the sample is 100 mg/dL (milligrams per deciliter), thevoltage application conditions are open (5 seconds)—250 mV, and theheight of the capillary is 150 μm;

FIG. 19B is a graph of the response current value under the sameconditions as in FIG. 19A, except that the height of the capillary is100 μm;

FIG. 19C is a graph of the response current value under the sameconditions as in FIG. 19A, except that the height of the capillary is 59μm;

FIG. 19D is a graph of the response current value under the sameconditions as in FIG. 19A, except that the height of the capillary is 33μm;

FIG. 20A is a graph of the response current value when 8 seconds haveelapsed in FIGS. 19A to 19D;

FIG. 20B is a graph of the variance in the response current value underthe various temperature conditions in FIG. 20A, using the responsecurrent value at 21° C. as a reference;

FIG. 21A is a graph of the response current value when the glucoseconcentration of the sample is 400 mg/dL (milligrams per deciliter), thevoltage application conditions are open (5 seconds)—250 mV, and theheight of the capillary is 150 μm;

FIG. 21B is a graph of the response current value under the sameconditions as in FIG. 21A, except that the height of the capillary is100 μm;

FIG. 21C is a graph of the response current value under the sameconditions as in FIG. 21A, except that the height of the capillary is 59μm;

FIG. 21D is a graph of the response current value under the sameconditions as in FIG. 21A, except that the height of the capillary is 33μm;

FIG. 22A is a graph of the response current value when 8 seconds haveelapsed in FIGS. 21A to 21D;

FIG. 22B is a graph of the variance in the response current value underthe various temperature conditions in FIG. 22A, using the responsecurrent value at 21° C. as a reference;

FIG. 23A is a graph of the response current value when the glucoseconcentration of the sample is 40 mg/dL (milligrams per deciliter), thevoltage application conditions are open (1.5 seconds)—250 mV, and theheight of the capillary is 150 μm;

FIG. 23B is a graph of the response current value under the sameconditions as in FIG. 23A, except that the height of the capillary is100 μm;

FIG. 23C is a graph of the response current value under the sameconditions as in FIG. 23A, except that the height of the capillary is 59μm;

FIG. 23D is a graph of the response current value under the sameconditions as in FIG. 23A, except that the height of the capillary is 33μm;

FIG. 24A is a graph of the response current value under the sameconditions as in FIG. 23A, except that the glucose concentration of thesample is 155 mg/dL;

FIG. 24B is a graph of the response current value under the sameconditions as in FIG. 24A, except that the height of the capillary is100 μm;

FIG. 24C is a graph of the response current value under the sameconditions as in FIG. 24A, except that the height of the capillary is 59μm;

FIG. 24D is a graph of the response current value under the sameconditions as in FIG. 24A, except that the height of the capillary is 33μm;

FIG. 25A is a graph of the response current value under the sameconditions as in FIG. 23A, except that the glucose concentration of thesample is 345 mg/dL;

FIG. 25B is a graph of the response current value under the sameconditions as in FIG. 25A, except that the height of the capillary is100 μm;

FIG. 25C is a graph of the response current value under the sameconditions as in FIG. 25A, except that the height of the capillary is 59μm;

FIG. 25D is a graph of the response current value under the sameconditions as in FIG. 25A, except that the height of the capillary is 33μm;

FIG. 26A is a graph of the response current value under the sameconditions as in FIG. 23A, except that the glucose concentration of thesample is 600 mg/dL;

FIG. 26B is a graph of the response current value under the sameconditions as in FIG. 26A, except that the height of the capillary is100 μm;

FIG. 26C is a graph of the response current value under the sameconditions as in FIG. 26A, except that the height of the capillary is 59μm;

FIG. 26D is a graph of the response current value under the sameconditions as in FIG. 26A, except that the height of the capillary is 33μm;

FIG. 27A is a graph of the relation between temperature, glucoseconcentration, and the current value when 5.5 seconds have elapsed (4seconds after the start of application of a voltage of 250 mV), when theheight of the capillary is 150 μm, on the basis of FIGS. 23A, 24A, 25A,and 26A;

FIG. 27B is a graph of the relation between temperature, glucoseconcentration, and the current value when 5.5 seconds have elapsed (4seconds after the start of application of a voltage of 250 mV), when theheight of the capillary is 100 μm, on the basis of FIGS. 23B, 24B, 25B,and 26B;

FIG. 27C is a graph of the relation between temperature, glucoseconcentration, and the current value when 5.5 seconds have elapsed (4seconds after the start of application of a voltage of 250 mV), when theheight of the capillary is 59 μm, on the basis of FIGS. 23C, 24C, 25C,and 26C;

FIG. 27D is a graph of the relation between temperature, glucoseconcentration, and the current value when 5.5 seconds have elapsed (4seconds after the start of application of a voltage of 250 mV), when theheight of the capillary is 33 μm, on the basis of FIGS. 23D, 24D, 25D,and 26D;

FIG. 28A is a graph of the response current value when the glucoseconcentration of the sample is 40 mg/dL, the voltage applicationconditions are open (3 seconds)—250 mV, and the height of the capillaryis 150 μm;

FIG. 28B is a graph of the response current value under the sameconditions as in FIG. 28A, except that the height of the capillary is100 μm;

FIG. 28C is a graph of the response current value under the sameconditions as in FIG. 28A, except that the height of the capillary is 59μm;

FIG. 28D is a graph of the response current value under the sameconditions as in FIG. 28A, except that the height of the capillary is 33μm;

FIG. 29A is a graph of the response current value under the sameconditions as in FIG. 28A (the voltage application conditions are open(3 seconds)—250 mV, and the height of the capillary is 150 μm), exceptthat the glucose concentration of the sample is 155 mg/dL;

FIG. 29B is a graph of the response current value under the sameconditions as in FIG. 29A, except that the height of the capillary is100 μm;

FIG. 29C is a graph of the response current value under the sameconditions as in FIG. 29A, except that the height of the capillary is 59μm;

FIG. 29D is a graph of the response current value under the sameconditions as in FIG. 29A, except that the height of the capillary is 33μm;

FIG. 30A is a graph of the response current value under the sameconditions as in FIG. 28A (the voltage application conditions are open(3 seconds)—250 mV, and the height of the capillary is 150 μm), exceptthat the glucose concentration of the sample is 345 mg/dL;

FIG. 30B is a graph of the response current value under the sameconditions as in FIG. 30A, except that the height of the capillary is100 μm;

FIG. 30C is a graph of the response current value under the sameconditions as in FIG. 30A, except that the height of the capillary is 59μm;

FIG. 30D is a graph of the response current value under the sameconditions as in FIG. 30A, except that the height of the capillary is 33μm;

FIG. 31A is a graph of the response current value under the sameconditions as in FIG. 28A (the voltage application conditions are open(3 seconds)—250 mV, and the height of the capillary is 150 μm), exceptthat the glucose concentration of the sample is 600 mg/dL;

FIG. 31B is a graph of the response current value under the sameconditions as in FIG. 31A, except that the height of the capillary is100 μm;

FIG. 31C is a graph of the response current value under the sameconditions as in FIG. 31A, except that the height of the capillary is 59μm;

FIG. 31D is a graph of the response current value under the sameconditions as in FIG. 31A, except that the height of the capillary is 33μm;

FIG. 32A is a graph of the relation between temperature, glucoseconcentration, and the current value when 7 seconds have elapsed (4seconds after the start of application of a voltage of 250 mV), when theheight of the capillary is 150 μm, on the basis of FIGS. 28A, 29A, 30A,and 31A;

FIG. 32B is a graph of the relation between temperature, glucoseconcentration, and the current value when 7 seconds have elapsed (4seconds after the start of application of a voltage of 250 mV), when theheight of the capillary is 100 μm, on the basis of FIGS. 28B, 29B, 30B,and 31B;

FIG. 32C is a graph of the relation between temperature, glucoseconcentration, and the current value when 7 seconds have elapsed (4seconds after the start of application of a voltage of 250 mV), when theheight of the capillary is 59 μm, on the basis of FIGS. 28C, 29C, 30C,and 31C;

FIG. 32D is a graph of the relation between temperature, glucoseconcentration, and the current value when 7 seconds have elapsed (4seconds after the start of application of a voltage of 250 mV), when theheight of the capillary is 33 μm, on the basis of FIGS. 28D, 29D, 30D,and 31D;

FIG. 33A is a graph of the response current value when the glucoseconcentration of the sample is 100 mg/dL, neither the application ofopen circuit voltage nor the application of low voltage is executed, theapplied voltage is 250 mV, and the height of the capillary is 150 μm;

FIG. 33B is a graph of the response current value under the sameconditions as in FIG. 33A, except that the height of the capillary is104 μm;

FIG. 33C is a graph of the response current value under the sameconditions as in FIG. 33A, except that the height of the capillary is 90μm;

FIG. 33D is a graph of the response current value under the sameconditions as in FIG. 33A, except that the height of the capillary is 82μm;

FIG. 34A is a graph of the response current value under the sameconditions as in FIG. 33A, except that the height of the capillary is 69μm;

FIG. 34B is a graph of the response current value under the sameconditions as in FIG. 33A, except that the height of the capillary is 59μm;

FIG. 34C is a graph of the response current value under the sameconditions as in FIG. 33A, except that the height of the capillary is 49μm;

FIG. 34D is a graph of the response current value under the sameconditions as in FIG. 33A, except that the height of the capillary is 33μm;

FIG. 35A is a graph of the response current value under the sameconditions as in FIG. 33A, except that the voltage applicationconditions are open (1 second)—250 mV;

FIG. 35B is a graph of the response current value under the sameconditions as in FIG. 35A, except that the height of the capillary is104 μm;

FIG. 35C is a graph of the response current value under the sameconditions as in FIG. 35A, except that the height of the capillary is 90μm;

FIG. 35D is a graph of the response current value under the sameconditions as in FIG. 35A, except that the height of the capillary is 82μm;

FIG. 36A is a graph of the response current value under the sameconditions as in FIG. 35A, except that the height of the capillary is 69μm;

FIG. 36B is a graph of the response current value under the sameconditions as in FIG. 35A, except that the height of the capillary is 59μm;

FIG. 36C is a graph of the response current value under the sameconditions as in FIG. 35A, except that the height of the capillary is 49μm;

FIG. 36D is a graph of the response current value under the sameconditions as in FIG. 35A, except that the height of the capillary is 33μm;

FIG. 37A is a graph of the response current value under the sameconditions as in FIG. 33A, except that the voltage applicationconditions are open (1.5 seconds)—250 mV;

FIG. 37B is a graph of the response current value under the sameconditions as in FIG. 37A, except that the height of the capillary is104 μm;

FIG. 37C is a graph of the response current value under the sameconditions as in FIG. 37A, except that the height of the capillary is 90μm;

FIG. 37D is a graph of the response current value under the sameconditions as in FIG. 37A, except that the height of the capillary is 82μm;

FIG. 38A is a graph of the response current value under the sameconditions as in FIG. 37A, except that the height of the capillary is 69μm;

FIG. 38B is a graph of the response current value under the sameconditions as in FIG. 37A, except that the height of the capillary is 59μm;

FIG. 38C is a graph of the response current value under the sameconditions as in FIG. 37A, except that the height of the capillary is 49μm;

FIG. 38D is a graph of the response current value under the sameconditions as in FIG. 37A, except that the height of the capillary is 33μm;

FIG. 39A is a graph of the response current value under the sameconditions as in FIG. 33A, except that the voltage applicationconditions are open (2 seconds)—250 mV;

FIG. 39B is a graph of the response current value under the sameconditions as in FIG. 39A, except that the height of the capillary is104 μm;

FIG. 39C is a graph of the response current value under the sameconditions as in FIG. 39A, except that the height of the capillary is 90μm;

FIG. 39D is a graph of the response current value under the sameconditions as in FIG. 39A, except that the height of the capillary is 82μm;

FIG. 40A is a graph of the response current value under the sameconditions as in FIG. 39A, except that the height of the capillary is 69μm;

FIG. 40B is a graph of the response current value under the sameconditions as in FIG. 39A, except that the height of the capillary is 59μm;

FIG. 40C is a graph of the response current value under the sameconditions as in FIG. 39A, except that the height of the capillary is 49μm;

FIG. 40D is a graph of the response current value under the sameconditions as in FIG. 39A, except that the height of the capillary is 33μm;

FIG. 41A is a graph of the response current value under the sameconditions as in FIG. 33A, except that the voltage applicationconditions are open (3 seconds)—250 mV;

FIG. 41B is a graph of the response current value under the sameconditions as in FIG. 41A, except that the height of the capillary is104 μm;

FIG. 41C is a graph of the response current value under the sameconditions as in FIG. 41A, except that the height of the capillary is 90μm;

FIG. 41D is a graph of the response current value under the sameconditions as in FIG. 41A, except that the height of the capillary is 82μm;

FIG. 42A is a graph of the response current value under the sameconditions as in FIG. 41A, except that the height of the capillary is 69μm;

FIG. 42B is a graph of the response current value under the sameconditions as in FIG. 41A, except that the height of the capillary is 59μm;

FIG. 42C is a graph of the response current value under the sameconditions as in FIG. 41A, except that the height of the capillary is 49μm;

FIG. 42D is a graph of the response current value under the sameconditions as in FIG. 41A, except that the height of the capillary is 33μm;

FIG. 43A is a graph of the response current value under the sameconditions as in FIG. 33A, except that the voltage applicationconditions are open (4 seconds)—250 mV;

FIG. 43B is a graph of the response current value under the sameconditions as in FIG. 43A, except that the height of the capillary is104 μm;

FIG. 43C is a graph of the response current value under the sameconditions as in FIG. 43A, except that the height of the capillary is 90μm;

FIG. 43D is a graph of the response current value under the sameconditions as in FIG. 43A, except that the height of the capillary is 82μm;

FIG. 44A is a graph of the response current value under the sameconditions as in FIG. 43A, except that the height of the capillary is 69μm;

FIG. 44B is a graph of the response current value under the sameconditions as in FIG. 43A, except that the height of the capillary is 59μm;

FIG. 44C is a graph of the response current value under the sameconditions as in FIG. 43A, except that the height of the capillary is 49μm;

FIG. 44D is a graph of the response current value under the sameconditions as in FIG. 43A, except that the height of the capillary is 33μm;

FIG. 45A is a graph of the response current value under the sameconditions as in FIG. 33A, except that the voltage applicationconditions are open (5 seconds)—250 mV;

FIG. 45B is a graph of the response current value under the sameconditions as in FIG. 45A, except that the height of the capillary is104 μm;

FIG. 45C is a graph of the response current value under the sameconditions as in FIG. 45A, except that the height of the capillary is 90μm;

FIG. 45D is a graph of the response current value under the sameconditions as in FIG. 45A, except that the height of the capillary is 82μm;

FIG. 46A is a graph of the response current value under the sameconditions as in FIG. 45A, except that the height of the capillary is 69μm;

FIG. 46B is a graph of the response current value under the sameconditions as in FIG. 45A, except that the height of the capillary is 59μm;

FIG. 46C is a graph of the response current value under the sameconditions as in FIG. 45A, except that the height of the capillary is 49μm;

FIG. 46D is a graph of the response current value under the sameconditions as in FIG. 45A, except that the height of the capillary is 33μm;

FIG. 47A is a graph of the variance in the response current valuesmeasured at 5° C., 14° C., 30° C., and 38° C. when the glucoseconcentration of the sample is 100 mg/dL, neither the application ofopen circuit voltage nor the application of low voltage is executed, theapplied voltage is 250 mV, and the height of the capillary is 104 μm,using the response current value at 21° C. as a reference (the varianceobtained under the same conditions, except that the height of thecapillary is 150 μm, is shown for comparison);

FIG. 47B is a graph of the variance in the response current value underthe same conditions as in FIG. 47A, except that the height of thecapillary is 90 μm (the variance obtained under the same conditions,except that the height of the capillary is 150 μm, is shown forcomparison);

FIG. 47C is a graph of the variance in the response current value underthe same conditions as in FIG. 47A, except that the height of thecapillary is 82 μm (the variance obtained under the same conditions,except that the height of the capillary is 150 μm, is shown forcomparison)

FIG. 47D is a graph of the variance in the response current value underthe same conditions as in FIG. 47A, except that the height of thecapillary is 69 μm (the variance obtained under the same conditions,except that the height of the capillary is 150 μm, is shown forcomparison)

FIG. 48A is a graph of the variance in the response current value underthe same conditions as in FIG. 47A, except that the height of thecapillary is 59 μm (the variance obtained under the same conditions,except that the height of the capillary is 150 μm, is shown forcomparison);

FIG. 48B is a graph of the variance in the response current value underthe same conditions as in FIG. 47A, except that the height of thecapillary is 49 μm (the variance obtained under the same conditions,except that the height of the capillary is 150 μm, is shown forcomparison);

FIG. 48C is a graph of the variance in the response current value underthe same conditions as in FIG. 47A, except that the height of thecapillary is 33 μm (the variance obtained under the same conditions,except that the height of the capillary is 150 μm, is shown forcomparison);

FIG. 49A is a graph of the variance in the response current valuesmeasured at 5° C., 14° C., 30° C., and 38° C. when the glucoseconcentration of the sample is 100 mg/dL, the voltage applicationconditions are open (2 seconds)—250 mV, and the height of the capillaryis 104 μm, using the response current value at 21° C. as a reference(the variance obtained under the same conditions, except that the heightof the capillary is 150 μm, is shown for comparison);

FIG. 49B is a graph of the variance in the response current value underthe same conditions as in FIG. 49A, except that the height of thecapillary is 90 μm (the variance obtained under the same conditions,except that the height of the capillary is 150 μm, is shown forcomparison);

FIG. 49C is a graph of the variance in the response current value underthe same conditions as in FIG. 49A, except that the height of thecapillary is 82 μm (the variance obtained under the same conditions,except that the height of the capillary is 150 μm, is shown forcomparison);

FIG. 49D is a graph of the variance in the response current value underthe same conditions as in FIG. 49A, except that the height of thecapillary is 69 μm (the variance obtained under the same conditions,except that the height of the capillary is 150 μm, is shown forcomparison);

FIG. 50A is a graph of the variance in the response current value underthe same conditions as in FIG. 49A, except that the height of thecapillary is 59 μm (the variance obtained under the same conditions,except that the height of the capillary is 150 μm, is shown forcomparison);

FIG. 50B is a graph of the variance in the response current value underthe same conditions as in FIG. 49A, except that the height of thecapillary is 49 μm (the variance obtained under the same conditions,except that the height of the capillary is 150 μm, is shown forcomparison);

FIG. 50C is a graph of the variance in the response current value underthe same conditions as in FIG. 49A, except that the height of thecapillary is 33 μm (the variance obtained under the same conditions,except that the height of the capillary is 150 μm, is shown forcomparison);

FIG. 51A is a graph of the variance in the response current values whenthe glucose concentration of the sample is 100 mg/dL, the voltageapplication conditions are open (3 seconds)—250 mV, and the height ofthe capillary is 150 μm;

FIG. 51B is a graph of the response current value under the sameconditions as in FIG. 51A, except that the height of the capillary is104 μm;

FIG. 51C is a graph of the response current value under the sameconditions as in FIG. 51A, except that the height of the capillary is 90μm;

FIG. 51D is a graph of the response current value under the sameconditions as in FIG. 51A, except that the height of the capillary is 82μm;

FIG. 52A is a graph of the response current value under the sameconditions as in FIG. 51A, except that the height of the capillary is 69μm;

FIG. 52B is a graph of the response current value under the sameconditions as in FIG. 51A, except that the height of the capillary is 59μm;

FIG. 52C is a graph of the response current value under the sameconditions as in FIG. 51A, except that the height of the capillary is 49μm;

FIG. 52D is a graph of the response current value under the sameconditions as in FIG. 51A, except that the height of the capillary is 33μm;

FIG. 53A is a graph of the response current value when the glucoseconcentration of the sample is 100 mg/dL, the voltage applicationconditions are 0 mV (3 seconds)—250 mV, and the height of the capillaryis 150 μm;

FIG. 53B is a graph of the response current value under the sameconditions as in FIG. 53A, except that the height of the capillary is104 μm;

FIG. 53C is a graph of the response current value under the sameconditions as in FIG. 53A, except that the height of the capillary is 90μm;

FIG. 53D is a graph of the response current value under the sameconditions as in FIG. 53A, except that the height of the capillary is 82μm;

FIG. 54A is a graph of the response current value under the sameconditions as in FIG. 53A, except that the height of the capillary is 69μm;

FIG. 54B is a graph of the response current value under the sameconditions as in FIG. 53A, except that the height of the capillary is 59μm;

FIG. 54C is a graph of the response current value under the sameconditions as in FIG. 53A, except that the height of the capillary is 49μm;

FIG. 54D is a graph of the response current value under the sameconditions as in FIG. 53A, except that the height of the capillary is 33μm;

FIG. 55A is a graph of the response current value when the glucoseconcentration of the sample is 100 mg/dL, the voltage applicationconditions are 250 mV (1 second)—open (2 seconds)—250 mV, and the heightof the capillary is 150 μm;

FIG. 55B is a graph of the response current value under the sameconditions as in FIG. 55A, except that the height of the capillary is104 μm;

FIG. 55C is a graph of the response current value under the sameconditions as in FIG. 55A, except that the height of the capillary is 90μm;

FIG. 55D is a graph of the response current value under the sameconditions as in FIG. 55A, except that the height of the capillary is 82μm;

FIG. 56A is a graph of the response current value under the sameconditions as in FIG. 55A, except that the height of the capillary is 69μm;

FIG. 56B is a graph of the response current value under the sameconditions as in FIG. 55A, except that the height of the capillary is 59μm;

FIG. 56C is a graph of the response current value under the sameconditions as in FIG. 55A, except that the height of the capillary is 49μm; and

FIG. 56D is a graph of the response current value under the sameconditions as in FIG. 55A, except that the height of the capillary is 33μm.

DESCRIPTION OF EMBODIMENTS

A biosensor system 100 featuring a sensor chip 200 pertaining to anembodiment of the present invention will now be described.

1. Configuration of Biosensor System

The biosensor system 100 pertaining to this embodiment is a system thatincludes a sensor for measuring the concentration of an analyte includedin a liquid sample. As shown in FIG. 1, the biosensor system 100 has ameasurement device 101 and the sensor chip 200.

The liquid sample is not limited to being any particular sample, and avariety of samples can be used, such as blood, perspiration, urine, andother such biologically derived liquid samples (biological samples);liquid samples that come from a river, the ocean, a lake, or anothersuch environment; and liquid samples that come from food. The biosensorsystem 100 is preferably applied to a biological sample, andparticularly to blood.

Nor is the analyte (the substance to be measured) limited to anyparticular substance, and the sensor chip 200 can accommodate any of avariety of substances, by changing the enzyme or the like in a reagentlayer 20 (discussed below). Examples of analytes in a blood sampleinclude substances excluding blood cells, such as glucose, albumin,lactic acid, bilirubin, and cholesterol.

The measurement device 101 has in its side wall a mounting opening 102,which is a rectangular hole. The sensor chip 200 can be connected in aremovable state to the mounting opening 102. A display section 103 thatdisplays measurement results is disposed in the approximate center ofone main face of the measurement device 101. The configuration of themeasurement device 101 will be discussed in detail below.

2. Sensor Chip

2-1. Configuration of Sensor Chip

The sensor chip 200 is a disposable sensor chip that is discarded aftera single use. As shown in FIGS. 2 and 3, the sensor chip 200 comprisesan insulated board 201, a spacer 202, and a cover 203. The cover 203 isdisposed on the insulated board 201 with the spacer 202 in between. Theinsulated board 201, the spacer 202, and the cover 203 are integratedadhesively, by heat fusion, or the like, for example.

The materials of the insulated board 201, the spacer 202, and the cover203 can be polyethylene terephthalate, polycarbonate, polyimide,polyethylene, polypropylene, polystyrene, polyvinyl chloride,polyoxymethylene, monomer cast nylon, polybutylene terephthalate,methacrylic resin, ABS resin, and other such resins, and glass.

The sensor chip 200 further comprises a capillary 40 (FIG. 3). Thecapillary 40 holds a liquid sample. The capillary 40 is constituted by acut-out 204 in the spacer 202. The capillary 40 has a shape that islonger in the long-side direction of the sensor chip 200. The capillary40 leads to the outside of the sensor chip 200 at one end of the spacer202 (the end on the left in FIGS. 2 and 3). In other words, the sensorchip 200 comprises an introduction port 17 that opens outward, and thecapillary 40 is connected to and communicates with the introduction port17. The volume of the liquid sample introduced into the capillary 40 is1 μL or less, for example.

Three electrodes 11 to 13 are provided on the surface of the insulatedboard 201. The electrode 11 is sometimes called a working electrode, theelectrode 12 a counter electrode, and the electrode 13 a detectingelectrode. A portion of each of the electrodes 11 to 13 is disposedwithin the capillary 40. The electrodes 11 to 13 are disposed so as tobe aligned in the order of the electrode 12, the electrode 11, theelectrode 12, and the electrode 13, from the introduction port 17 towardthe interior of the capillary 40. That is, in FIG. 5A, the electrodesare disposed so as to be opposite each other in the planar direction ofthe insulated board 201.

However, as shown in FIG. 5B, the electrode 11, the electrode 12, andthe electrode 13 may be disposed three-dimensionally. For instance, theelectrode 12 may be provided at a location opposite the capillary 40 onthe lower face of the cover 203, and the electrode 11 and the electrode13 may be provided on the insulated board 201.

There are no particular restrictions on the number of electrodes 11 to13 used in the sensor chip 200. The number of each of the electrodes maybe two or more.

The material of the electrodes 11 to 13 may be palladium, platinum,gold, silver, titanium, copper, nickel, carbon, or any other knownconductive material.

Also, the electrodes 11 to 13 are lined to leads 110, 120, and 130,respectively. The leads 110, 120, and 130 are provided on the insulatedboard 201. One end of the insulated board 201 is not covered by thespacer 202 and the cover 203. One end of the leads 110, 120, and 130 isnot covered on the insulated board 201, and is exposed outside thesensor chip 200. The measurement device 101 applies voltage to theelectrodes 11 to 13 via the leads 110, 120, and 130.

An air vent 16 is provided to the cover 203 at a location facing theinner part of the cut-out 204 (the opposite side from the introductionport 17) that forms the capillary 40. Because the air vent 16 isprovided, the liquid sample introduced into the capillary 40 flows undercapillary action and in rate-limiting fashion to a detector constitutedby the electrodes 11 to 13 and the reagent layer 20. Thus, the air vent16 ensures the deposition of a blood sample (biological sample), andimproves measurement stability.

Also, the faces on the inside the capillary 40 may be given ahydrophilic treatment or formed from a hydrophilic material. Thisfacilitates the deposition (intake) of the liquid sample and improvesreliability.

The reagent layer 20 is placed on the electrodes 11 to 13 between theinsulated board 201 and the spacer 202.

The reagent layer 20 is formed by precoating the insulated board 201with a reagent that includes an electrolyte. The reagent layer 20 isformed so as to cover the overlapping portion of the electrodes 11, 12,and 13 on the insulated board 201. The reagent layer 20 contains anelectron-transfer mediator (hereinafter referred to simply as a“mediator”) and a redox enzyme in which the analyte in the liquid sampleserves as the substrate.

A redox enzyme in which the analyte serves as the substrate can be usedfavorably as the enzyme. Examples of this enzyme include glucose oxidaseand glucose dehydrogenase when the analyte is glucose; lactic acidoxidase and lactic acid dehydrogenase when the analyte is lactic acid;cholesterol esterase and cholesterol oxidase when the analyte ischolesterol; and bilirubin oxidase when the analyte is bilirubin. Otherexamples of analyte include triglyceride and uric acid.

The mediator is a substance having the function of transferringelectrons produced by an enzyme reaction to the electrodes. One or moretypes of mediator selected from the group consisting of potassiumferricyanide, p-benzoquinone, p-benzoquinone derivatives, oxide-typephenazine methosulfate, methylene blue, ferricinium, and ferriciniumderivatives can be used favorably, for example.

The amount of redox enzyme in the reagent layer will vary with the typeof enzyme and so forth, in general, 0.01 to 100 units (U) is favorable,with 0.05 to 10 U being preferable, and 0.1 to 5 U being even better.

The reagent layer 20 may contain a water-soluble polymer compound inorder to improve the moldability of the reagent layer. Thiswater-soluble polymer compound may be one or more types selected fromamong carboxymethyl cellulose and salts thereof, hydroxyethyl cellulose,hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, ethylhydroxyethyl cellulose, carboxyethyl cellulose, and salts thereof,polyvinyl alcohol, polyvinylpyrrolidone, polylysine, and other suchpolyamino acids; polystyrenesulfonic acid and salts thereof; gelatin andderivatives thereof; polyacrylic acid and salts thereof; polymethacrylicacid and salts thereof; starch and derivatives thereof; maleic anhydridepolymers and salts thereof, and agarose gel and derivatives thereof.

2-2. Height of Capillary 40

The system inside the capillary 40 into which the liquid sample isintroduced is a diffusion system including a liquid and a diffusant(analyte and mediator, etc.) The liquid here could also be called adiffusion medium or a dispersion medium.

The diffusion distance d of the various diffusants in the liquid isexpressed by the following formula (1).

[First Mathematical Formula]

d=√{square root over (zDt)}  (1)

z: a constant

D: diffusion coefficient

t: time

The constant z is an arbitrarily selected value. The constant z can varywith the experiment conditions, and can vary according to the definitionof the distribution of the distance over which a diffusant is diffused.The constant z is generally set to a range of 1≦z≦4. More specifically,the constant z may be defined as 1, 2, π, or 4. In the field ofelectrochemistry, z=π is sometimes used as an example, so we will usez=π in the following description.

The diffusion coefficient D is expressed by a Stokes-Einstein relation(the following formula (2)).

$\begin{matrix}\left\lbrack {{Second}\mspace{14mu} {Mathematical}\mspace{14mu} {Formula}} \right\rbrack & \; \\{D = \frac{k\; T}{6\; \pi \; \mu \; r}} & (2)\end{matrix}$

k: Boltzmann constant

T: absolute temperature

μ: viscosity

r: radius of diffused molecules

Thus, based on Formulas 1 and 2, the diffusion distance d is expressedby the following formula (3).

$\begin{matrix}\left\lbrack {{Third}\mspace{14mu} {Mathematical}\mspace{14mu} {Formula}} \right\rbrack & \; \\{d = \sqrt{\frac{tKT}{6\; \mu \; r}}} & (3)\end{matrix}$

That is, in general, when the temperature rises, the diffusion distanceincreases.

Furthermore, in a system in which the viscosity μ is dependent ontemperature, the viscosity μ is expressed by an Andrade formula (4).

$\begin{matrix}\left\lbrack {{Fourth}\mspace{14mu} {Mathematical}\mspace{14mu} {Formula}} \right\rbrack & \; \\{\mu = {A\; {\exp \left( \frac{E}{RT} \right)}}} & (4)\end{matrix}$

A: proportional constant

E: fluid activation energy

R: gas constant

T: absolute temperature

If we plug the above-mentioned Formula 3 into the above-mentionedFormula 4, we obtain the following formula (5).

$\begin{matrix}\left\lbrack {{Fifth}\mspace{14mu} {Mathematical}\mspace{14mu} {Formula}} \right\rbrack & \; \\\begin{matrix}{d = \sqrt{\frac{tKT}{6\; \mu \; r}}} \\{= \sqrt{\frac{t\; k\; T}{6\; r\; A\; \exp \mspace{14mu} \left( \frac{E}{RT} \right)}}}\end{matrix} & (5)\end{matrix}$

Here, if we assume that:

$\begin{matrix}\left\lbrack {{Sixth}\mspace{14mu} {Mathematical}\mspace{14mu} {Formula}} \right\rbrack & \; \\{\sqrt{\frac{k}{6\; r\; A}} = B} & (6)\end{matrix}$

B: constant term

then the diffusion distance d is expressed by the following formula (7).

$\begin{matrix}\left\lbrack {{Seventh}\mspace{14mu} {Mathematical}\mspace{14mu} {Formula}} \right\rbrack & \; \\{d = {B\sqrt{\frac{t\; T}{\exp \mspace{14mu} \left( \frac{E}{RT} \right)}}}} & (7)\end{matrix}$

In the above-mentioned Formula 7, when the temperature T rises, theexp(E/RT) of the denominator decreases, so there is a further increasein the diffusion distance d.

In general, the fluid activation energy E is large in a high-viscosityliquid, so the viscosity μ is susceptible to the effect of thetemperature T. As a result, the higher is the viscosity of the liquidsample, the more susceptible are the diffusion coefficient D and thediffusion distance d to the effect of the temperature T. For example,when the temperature T rises, the viscosity μ decreases and thediffusion coefficient D and the diffusion distance d increase.

As shown in FIG. 4, an analyte 51 diffuses into the interior of thereagent layer 20, and transfers electrons through an enzyme reaction toa mediator 54. The mediator 54 diffuses to the working electrode 11.

If we let D_(A) be the diffusion constant of the analyte 51, and lett_(A) be the diffusion time it takes for the analyte to transferelectrons to the mediator, then the diffusion distance d_(A) of theanalyte 51 is expressed by the following formula (8). The diffusion timet_(A) is an arbitrarily set numerical value.

[Eighth Mathematical Formula]

d _(A)=√{square root over (πD _(A) t _(A))}  (8)

Similarly, if we let D_(M) be the diffusion constant of the mediator 54,and let t_(M) be the diffusion time it takes for the mediator 54 totransfer electrons to the working electrode 11, then the diffusiondistance d_(M) of the mediator 54 is expressed by the following formula(9). The diffusion time t_(M) is also an arbitrarily set numericalvalue.

[Ninth Mathematical Formula]

d _(M)=√{square root over (D _(M) t _(M))}  (9)

Based on the above-mentioned Formulas 8 and 9, the total diffusiondistance d_(T) until the analyte 51 is detected as a current response isexpressed by the following formula (10).

[Tenth Mathematical Formula]

d _(T)=√{square root over (πD _(A) T _(A))}+√{square root over (D _(M) t_(M))}  (10)

The time until the analyte 51 is detected as a current response can alsobe expressed by (t_(A)+t_(M)). Thus, if we let t_(mes) be themeasurement time, the analyte 51 will be detected within the measurementtime_(mes) if the measurement time t_(mes) satisfies the relationt_(mes)≧t_(A)+t_(M). That is, in this case the maximum value of(t_(A)+t_(M)) is t_(mes).

If the diffusion time t_(M) of the mediator 54 is sufficiently shortwith respect to the diffusion time t_(A) of the analyte 51, the systemcan be considered to be one in which only the analyte 51 moves onephase. Here, the maximum value & of the total diffusion distance d_(T)is expressed by the following formula (11).

$\begin{matrix}\left\lbrack {{Eleventh}\mspace{14mu} {Mathematical}\mspace{14mu} {Formula}} \right\rbrack & \; \\\begin{matrix}{d_{L} = \sqrt{\pi \; D_{A}t_{A}}} \\{= \sqrt{\pi \; D_{A}t_{mes}}}\end{matrix} & (11)\end{matrix}$

Since an enzyme is necessary for electron acceptance between the analyte51 and the mediator 54, the diffusion distance d_(M) of the mediator 54that has taken electrons is equal to the distance that the enzyme hasdiffused from the working electrode 11. In general, the diffusionconstant of an enzyme is far smaller than the diffusion coefficientD_(A) of the analyte 51 and the diffusion coefficient D_(M) of themediator 54. Accordingly, the enzyme can be considered to be in a stateof having stopped near the working electrode 11. Also, since thediffusion coefficient D_(M) of the mediator 54 is extremely short, thediffusion time t_(M) of the mediator can generally be ignored.

On the other hand, when the diffusion time t_(M) of the mediator islong, the diffusion system is considered to be one in which two kinds ofdiffusant move one phase. The maximum value & of the total diffusiondistance d_(T) is derived when the formula t_(mes)=t_(A)+t_(M) issatisfied in the above-mentioned Formula 10.

Furthermore, when the liquid sample is separated into a plurality ofphases, such as when a membrane filter is provided over the reagentlayer 20, the diffusion system inside the capillary 40 will be a systemin which the diffusant moves a plurality of phases (n number of phases).Thus, if the diffusion time t_(M) of the mediator 54 is sufficientlyshort with respect to the diffusion time t_(A) of the analyte 51, thediffusion constant and diffusion time of the analyte 51 are defined foreach phase, and the total diffusion distance d_(T) is expressed by thefollowing formula (12).

$\begin{matrix}\left\lbrack {{Twelfth}\mspace{14mu} {Mathematical}\mspace{14mu} {Formula}} \right\rbrack & \; \\{d_{T} = {\sum\limits_{k = 1}^{n}\; \sqrt{\pi \; D_{k}t_{k}}}} & (12)\end{matrix}$

(where n is an integer of 2 or more)

Here, if the following formula (13) is satisfied, the maximum valued_(L) of the total diffusion distance d_(T) can be derived.

$\begin{matrix}\left\lbrack {{Thirteenth}\mspace{14mu} {Mathematical}\mspace{14mu} {Formula}} \right\rbrack & \; \\{t_{mes} = {\sum\limits_{k = 1}^{n}\; t_{k}}} & (13)\end{matrix}$

On the other hand, if the diffusion time t_(M) of the mediator is long,the diffusion system can be considered to be one in which two kinds ofdiffusant move a plurality of phases (n number of phases), the diffusionconstant and diffusion time of the analyte 51 and the mediator 54 aredefined for each phase, and the total diffusion distance d_(T) isexpressed by the following formula (14).

$\begin{matrix}\left\lbrack {{Fourteenth}\mspace{14mu} {Mathematical}\mspace{14mu} {Formula}} \right\rbrack & \; \\{d_{T} = {{\sum\limits_{k = 1}^{n}\; \sqrt{\pi \; D_{k}t_{k}}} + {\sum\limits_{j = 1}^{n}\; \sqrt{\pi \; D_{j}t_{j}}}}} & (14)\end{matrix}$

(where n is an integer of 2 or more)

Here, if the following formula (15) is satisfied, the maximum valued_(L) of the total diffusion distance d_(T) can be derived.

$\begin{matrix}\left\lbrack {{Fifteenth}\mspace{14mu} {Mathematical}\mspace{14mu} {Formula}} \right\rbrack & \; \\{t_{mes} = {{\sum\limits_{k = 1}^{n}\; t_{k}} + {\sum\limits_{j = 1}^{n}\; t_{j}}}} & (15)\end{matrix}$

The formulas given above are formulas applied to systems of infinitediffusion. A system of infinite diffusion corresponds to when the heightH of the capillary 40 is set high. On the other hand, when the height His set low, the diffusion system inside the capillary 40 becomes asystem of finite diffusion. In this case, the range over which theanalyte 51 can diffuse is limited by the height H.

Whether or not the height H is greater than the maximum value d_(L) ofthe total diffusion distance d_(T) becomes a boundary for the diffusionsystem inside the capillary 40 will be of finite diffusion or infinitediffusion. Specifically, when the diffusion time t_(M) of the mediator54 is sufficiently short with respect to the diffusion time t_(A) of theanalyte 51, the inside of the capillary 40 becomes a system of finitediffusion when the height H satisfies the following formula (16).

[Sixteenth Mathematical Formula]

√{square root over (πD _(A) t _(A))}=√{square root over (πD _(A) t_(mes))}>H  (16)

On the other hand, when the diffusion time t_(M) of the mediator 54 islong, and the diffusion system inside the capillary 40 is one in whichtwo kinds of diffusant move one phase, then the diffusion system insidethe capillary 40 will be a system of finite diffusion when the height Hsatisfies the following formula (17) under a condition of t_(mes)=t_(A)t_(M).

[Seventeenth Mathematical Formula]

√{square root over (πD _(A) t _(A))}+√{square root over (πD _(M) t_(M))}>H  (17)

Also, when the system inside the capillary 40 is one in which thediffusion time t_(M) of the mediator 54 is sufficiently short withrespect to the diffusion time t_(A) of the analyte 51, and the analyte51 moves a plurality of phases (n number of phases), then the diffusionsystem inside the capillary 40 will be a system of finite diffusion whenthe height H satisfies the following formula (18) under the condition ofthe above-mentioned Formula 13.

$\begin{matrix}\left\lbrack {{Eighteenth}\mspace{14mu} {Mathematical}\mspace{14mu} {Formula}} \right\rbrack & \; \\{{\sum\limits_{k = 1}^{n}\sqrt{\pi \; D_{k}t_{k}}} > H} & (18)\end{matrix}$

Furthermore, when the system inside the capillary 40 is one in which thediffusion time t_(M) of the mediator 54 is long, and two kinds ofdiffusant move a plurality of phases (n number of phases), then thediffusion system inside the capillary 40 will be a system of finitediffusion when the height H satisfies the following formula (19) underthe condition of the above-mentioned Formula 15.

$\begin{matrix}\left\lbrack {{Nineteenth}\mspace{14mu} {Mathematical}\mspace{14mu} {Formula}} \right\rbrack & \; \\{{{\sum\limits_{k = 1}^{n}\sqrt{\pi \; D_{k}t_{k}}} + {\sum\limits_{j = 1}^{n}\; \sqrt{\pi \; D_{j}t_{j}}}} > H} & (19)\end{matrix}$

The diffusion coefficient D is found using experiment variables andcurrent values and using polarography, a rotating disk electrode method,a potential sweep method, a potential step method, or another suchmethod in the field of electrochemistry. The diffusion coefficient D isalso found by a measurement method based on something other thanelectrochemistry, such as a Taylor dispersion method, a nuclear magneticresonance-oblique magnetic field method, or the like. In general, thediffusion coefficient of the analyte is 1×10⁻⁵ cm²·s⁻¹ or less, and thediffusion coefficient of the mediator is also 1×10⁻⁵ cm²·s⁻¹ or less.

As shown in FIG. 5A, the height H of the capillary 40 is, in morespecific terms, the distance from the working electrode 11 to the innerface of the cover 203 (the opposite face from the working electrode 11).That is, the height H may be the thickness of the spacer 202, or may bea value obtained by adding the thickness of the reagent layer 20 to thethickness of the spacer 202.

The height H is set so that the diffusion system inside the capillary 40will be a system of finite diffusion. The range of the height H here isas described through reference to Formulas 16 to 19 above. As discussedabove, the diffusion distance d is a function of temperature. Thus, theheight H is preferably set to be less than the maximum value d_(L) ofthe total diffusion distance d_(T) found at the upper limit of themeasurement guaranteed temperature of the biosensor system 100. Thussetting the height H has the effect of minimizing the variance in themeasurement results at high temperature with the biosensor system 100.More preferably, the height H is set to be less than the maximum value &of the total diffusion distance d_(T) found at the lower limit of themeasurement guaranteed temperature of the biosensor system 100. Thussetting the height H has the advantage that concentration can bemeasured over a wide range of temperatures using a single calibrationcurve with the biosensor system 100.

The layout of the working electrode 11 and the counter electrode 12 isnot limited to one in which they are opposite each other in the planardirection of the insulated board 201 as in FIG. 5A. For instance, theworking electrode 11 and the counter electrode 12 may be disposedopposite each other in the height H direction of the capillary 40. Aspecific configuration is shown in FIG. 5B. In the example shown in FIG.5B, the working electrode 11 is disposed on the insulated board 201, andthe counter electrode 12 is disposed on the face of the cover 203 thatis opposite the insulated board 201. With this layout, the height H isthe distance between the working electrode 11 and the counter electrode12. Again with the layout in FIG. 5B, the height H is preferably withinthe range discussed above.

With the configurations in both FIG. 5A and FIG. 5B, the overall heightof the capillary 40 does not have to be within the above-mentionedrange, as long as the distance from the working electrode 11 to theportion opposite the working electrode 11 (the cover 203 in FIG. 5A, andthe counter electrode 12 in FIG. 5B) is within the above-mentionedrange.

3. Measurement Device 101

As shown in FIG. 6, the measurement device 101 has a control circuit 300in addition to the constitution discussed above. The control circuit 300applies voltage between at least two electrodes selected from among theelectrodes 11 to 13 of the sensor chip 200 (see FIGS. 2 and 3).

More specifically, as shown in FIG. 6, the control circuit 300 has threeconnectors 301 a, 301 b, and 301 c, a switching circuit 302, acurrent/voltage conversion circuit 303, an analog/digital conversioncircuit (hereinafter referred to as an A/D conversion circuit) 304, areference voltage source 305, and a computer 306. The control circuit300 can switch the voltage applied to one electrode, via the switchingcircuit 302, so that this electrode can be used as a positive ornegative pole.

As shown in FIG. 6, the connectors 301 a, 301 b, and 301 c are connectedto the counter electrode 12, the detection electrode 13, and the workingelectrode 11, respectively, in a state in which the sensor chip 200 isinserted into the mounting opening 102.

The switching circuit 302 can switch the electrode connected to thereference voltage source 305, and can switch the amount of voltageapplied to the electrodes.

The current/voltage conversion circuit 303 receives from the computer306 a signal directing the acquisition of a current value, and therebyconverts the amount of current flowing between two electrodes connectedto the current/voltage conversion circuit 303 into a voltage value. Theconverted voltage value is converted by the A/D conversion circuit 304into a digital value, inputted to the computer 306, and stored in thememory of the computer 306.

The computer 306 comprises a known central processing unit (CPU) and astorage unit. Examples of the storage unit include a HDD (hard diskdrive), ROM (read only memory), and RAM (random access memory). Thestorage unit stores a calibration curve that correlates the analyteconcentration in a blood sample with the current value between theworking electrode 11 and the counter electrode 12. The computer 306 canrefer to the calibration curve to compute the concentration of theanalyte in the blood sample.

Also, in addition to having a function of calculating the concentrationof analyte as mentioned above, the computer 306 also controls theswitching circuit 302, takes input from the A/D conversion circuit 304,controls the voltage of the reference voltage source 305, controls thetiming of voltage application during concentration measurement, measuresthe application duration, etc. (timer function), outputs display data tothe display section 103, and communicates with external devices, andtherefore controls the entire measurement device.

The various functions of the computer 306 can be realized by the CPU byreading and executing programs held in the storage unit.

4. Measurement of Analyte Concentration

When the sensor chip 200 is used, the user deposits a liquid sample atthe introduction port 17. For example, when the biosensor system 100 isused to measure a glucose value, the user pricks his finger, hand, arm,or the like, squeezes out a small amount of blood, and deposits thisblood as a liquid sample for measurement.

The liquid sample deposited at the introduction port 17 moves bycapillary action through the capillary 40 toward the back of the sensorchip 200, and reach the electrodes 11 to 13.

The measurement of analyte concentration performed by the biosensorsystem 100 will be described.

The operation shown in FIG. 7 begins when the sensor chip 200 is mountedin the mounting opening 102 of the measurement device 101. First, thedetection electrode 13 is connected to the current/voltage conversioncircuit 303 via the connector 301 b by the switching circuit 302 at acommand from the CPU of the computer 306, and the counter electrode 12is connected to the reference voltage source 305 via the connector 301a. After this, a specific voltage is applied between the two electrodesat a command from the CPU (step S11). This voltage is preferably 0.01 to2.0 V, and more preferably 0.1 to 1.0 V, and even more preferably 0.2 to0.5 V, when the detection electrode 13 is a positive pole and thecounter electrode 12 is a negative pole. This voltage is applied fromthe point when the sensor chip is inserted into the measurement device101 until the blood sample is introduced deep into the capillary 40.

When the blood sample is introduced from the introduction port 17 of thesensor chip 200 into the capillary 40, current flows between thedetection electrode 13 and the counter electrode 12. The CPU identifiesthe amount of increase in current per unit of time before and after theblood sample is introduced, and thereby detects that the capillary 40has been filled with the blood sample. The value of this current isconverted into a voltage value by the current/voltage conversion circuit303, after which it is converted into a digital value by the A/Dconversion circuit 304, and inputted to the CPU. The CPU detects thatthe blood sample has been introduced deep into the capillary on thebasis of this digital value.

When a sample is thus detected (Yes in step S12), step S13 is executed.Specifically, at a command from the CPU of the computer 306, theswitching circuit 302 disconnects the detection electrode 13 from thecurrent/voltage conversion circuit 303, connects the working electrode11 and the reference voltage source 305, and connects the counterelectrode 12 and the current/voltage conversion circuit 303. Morespecifically, the working electrode 11 is connected to thecurrent/voltage conversion circuit 303 via the connector 301 c, and thecounter electrode 12 is connected to the reference voltage source 305via the connector 301 a. An open circuit voltage is then applied betweenthe working electrode 11 and the counter electrode 12. The phrase “opencircuit voltage is applied” may be restated as “the voltage applicationis switched off”

As shown in FIG. 10A, the application time T₁ of the open circuitvoltage in step S13 is not limited to any specific value, as long as theeffect of temperature on the concentration measurement results can bereduced. The time T₁ is set to 0.5 to 15 seconds, for example, andpreferably 1 to 10 seconds, and more preferably 1 to 5 seconds, and evenmore preferably about 2 to 3 seconds.

Next, a measurement voltage V_(mes) is applied between the workingelectrode 11 and the counter electrode 12 under the control of thecomputer 306 (step S14). The amount of measurement voltage V_(mes)applied here can be varied according to the type of mediator and thetype of analyte being measured.

When the measurement voltage V_(mes) is applied, the value of thecurrent flowing between the working electrode 11 and the counterelectrode 12 is acquired (step S15). A signal directing the acquisitionof a current value is sent from the CPU of the computer 306 to thecurrent/voltage conversion circuit 303. The value of the current thatflows between the electrodes as a result of the application of themeasurement voltage V_(mes) is converted by the current/voltageconversion circuit 303 into a voltage value. After this, the convertedvoltage value is converted by the A/D conversion circuit 304 into adigital value and inputted to the CPU, then held in the memory of thecomputer 306. In this way, the current value at the time of measurementvoltage V_(mes) application is acquired in a state of having beenconverted into a digital voltage value.

The computer 306 calculates the concentration of analyte on the basis ofthe above-mentioned calibration curve and the digital value thus stored(step S16).

The effect of thus applying open circuit voltage prior to theapplication of the measurement voltage V_(mes) is that the concentrationmeasurement results are less likely to be affected by temperature.

In the above embodiment, a calibration curve was used for concentrationcalculation, but a table in which voltage values and concentration arecorrelated may be used in place of a calibration curve.

5. Other Embodiments—1

Step S13 in FIG. 7 is merely an example of processing that reduces theeffect of temperature on concentration measurement results. Thus, stepS13 can be replaced by some other processing. The open circuit voltageis an example of voltage that allows electrons to be accumulated in amediator, but any other voltage with which the effect of electronaccumulation can be obtained may be applied instead of an open circuitvoltage.

For example, as shown in FIG. 8, a step S23 may be executed instead ofstep S13. In step S23, a voltage that is lower than the measurementvoltage V_(mes) is applied between the working electrode 11 and thecounter electrode 12.

The “voltage that is lower than the measurement voltage V_(mes)” may beany voltage with which electrons can be accumulated in a mediator. Forinstance, when the measurement voltage V_(mes) is a voltage withpositive polarity, the voltage applied in step S23 may be voltage withpositive polarity (FIG. 10B), or may be 0 V (FIG. 10C), or may bevoltage with an inverse polarity, that is, a negative polarity (FIG.10D). More specifically, when the measurement voltage V_(mes) is 250 mV,the voltage applied in step S23 may be set to about −200 to 150 mV.

A state in which “electrons are accumulated in a mediator” means a statein which no electrons are transferred from the mediator to theelectrodes, or very few are transferred.

6. Other Embodiments—2

Steps S13 and S23 should be executed prior to the acquisition of acurrent value (steps S15 and S25). Another voltage application step maybe executed before or after steps S13 and S23.

For example, as shown in FIG. 9, another open circuit voltageapplication step S33 may be executed prior to a step S34 thatcorresponds to the above-mentioned step S23. There are no particularrestrictions on the amount of applied voltage in this step S33 (whichcould be called a third voltage application step), and may be largerthan the measurement voltage V_(mes).

In addition to the embodiment shown in FIG. 9, voltage may be applied inthe following combinations and orders.

-   -   (1) Third voltage application step/open circuit voltage        application step/measurement voltage application step    -   (2) Third voltage application step/open circuit voltage        application step/low voltage application step/measurement        voltage application step    -   (3) Open circuit voltage application step/low voltage        application step/measurement voltage application step

In all of these combinations, the low voltage application step mayinclude two or more voltage application steps of mutually differentvoltage values. Also, in all of these combinations, the open circuitvoltage application step and the low voltage application step may beswitched around.

The time “0” in FIGS. 10A to 10D may the point at which the introductionof a sample is detected, or may be the point at which a specific lengthof time has elapsed since this detection. Also, the duration of applyingthe open circuit voltage, the duration of applying the low voltage, orthe combined duration thereof is preferably 0.5 to 10 seconds. Forexample, it may be set to about 2 to 5 seconds.

As is clear from the description of the above embodiments, the computer306 and the reference voltage source 305 function as a first voltageapplicator for applying the measurement voltage V_(mes) (first voltage)between the working electrode 11 and the counter electrode 12, and asecond voltage applicator for applying a second voltage (open circuitvoltage, low voltage) prior to the application of the first voltage.

Furthermore, in the above embodiments, a single reference voltage source305 applies different voltages to the electrodes under the control ofthe computer 306, but in another constitution, the measurement device101 may have two or more voltage sources.

Also, the computer 306 functions as a concentration measurement sectionfor measuring the concentration of analyte.

7. Summary

The constitutions discussed in the different sections above can bevariously combined. Specifically, the embodiments given above can berephrased as follows.

1)

A biosensor system with which the concentration of an analyte in aliquid sample is measured using a redox enzyme and an electron-transfermediator, said biosensor system comprising a sensor chip comprising acapillary into which a liquid sample is introduced, whose height is lessthan the maximum value of the sum of the diffusion distance of theelectron-transfer mediator and the diffusion distance of the analyte atthe upper limit of the measurement guaranteed temperature of thebiosensor system, a plurality of electrodes disposed within thecapillary, and a reagent layer that is disposed within the capillary andincludes the electron-transfer mediator; a first voltage applicator thatapplies a first voltage to the electrodes; a concentration measurementsection that measures the concentration of the analyte on the basis ofthe value of the current flowing through the liquid sample during thefirst voltage application; and a second voltage applicator that appliesa second voltage to the electrodes prior to the application of the firstvoltage, so that the effect of the temperature of the liquid sample onthe measurement results of the concentration measurement section will bediminished.

2)

The biosensor system according to 1) above, wherein the height of thecapillary of the sensor chip is less than the maximum value of the sumof the diffusion distance of the electron-transfer mediator and thediffusion distance of the analyte, found from the lower limit of themeasurement guaranteed temperature of the biosensor system.

3)

The biosensor system according to 1) or 2) above, wherein the height ofthe capillary of the sensor chip is set on the basis of the diffusiondistances of the electron-transfer mediator and the analyte, eachexpressed by the following formula (i):

[First Mathematical Formula]

d=√{square root over (zDt)}  (i)

(where d is the diffusion distance, z is an arbitrarily selectedconstant, D is a diffusion coefficient, and t is time).

4)

The biosensor system according to 3) above,

wherein the constant z in the above Formula (i) satisfies 1≦z≦4.

5)

The biosensor system according to any of 1) to 4) above, wherein thesecond voltage applicator accumulates electrons in the electron-transfermediator by applying the second voltage.

6)

The biosensor system according to any of 1) to 5) above, wherein thesecond voltage applicator applies an open circuit voltage as the secondvoltage.

7)

The biosensor system according to any of 1) to 6) above, wherein thefirst voltage applicator applies voltage of positive polarity as thefirst voltage, and the second voltage applicator applies voltage that islower than the first voltage as the second voltage.

8)

The biosensor system according to any of Claims 1) to 7) above, whereinthe concentration measurement section has a calibration curve or tablethat correlates the current value and the analyte concentration, andcalculates the analyte concentration on the basis of the samecalibration curve or table even if the temperature of the liquid sampleshould fluctuate.

9)

The biosensor system according to any of 1) to 8) above, wherein theconcentration measurement section measures the analyte concentration onthe basis of the current value at a point when no more than 10 secondshave elapsed since the start of the application of the second voltage,and the height of the capillary of the sensor chip is no more than 90μm.

10)

The biosensor system according to any of 1) to 9), wherein theelectrodes are disposed on two faces that are mutually opposite in theheight direction of the capillary.

11)

A method for measuring the concentration of an analyte in a liquidsample using a redox enzyme or an electron-transfer mediator, which isexecuted by a biosensor system having a sensor chip comprising acapillary into which a liquid sample is introduced, whose height is lessthan the maximum value of the sum of the diffusion distance of theelectron-transfer mediator and the diffusion distance of the analyte atthe upper limit of the measurement guaranteed temperature of thebiosensor system, a plurality of electrodes disposed within thecapillary, and a reagent layer that is disposed within the capillary andincludes the electron-transfer mediator, said measurement methodcomprising a first voltage application step of applying a first voltageto the electrodes, a current detection step of detecting the value ofcurrent flowing through the liquid sample during the application of thefirst voltage, a concentration measurement step of measuring theconcentration of the analyte on the basis of the current value, and asecond voltage application step of applying a second voltage to theelectrodes prior to the detection of the current value, so that thetemperature of the liquid sample will have less effect on themeasurement results of the concentration measurement section.

12)

The measurement method according to 11) above, wherein the secondvoltage is set such that electrons will be accumulated in theelectron-transfer mediator by the application of the second voltage.

13)

The measurement method according to 11) or 12), wherein the averagemolecular weight is an open circuit voltage.

14)

The measurement method according to any of 11) to 13) above, wherein thefirst voltage is a voltage of positive polarity, and the second voltageis a voltage that is lower than the first voltage.

15)

The measurement method according to any of 11) to 14) above, wherein theconcentration measurement step includes a calculation step of using acalibration curve or table that correlates the current value and theanalyte concentration, and calculating the analyte concentration on thebasis of the same calibration curve or table even if the temperature ofthe liquid sample should fluctuate.

16)

The measurement method according to any of 11) to 15) above, wherein theheight of the capillary of the sensor chip is no more than 90 μm, and inthe concentration measurement step, the concentration of the analyte ismeasured on the basis of the current value at a point when no more than10 seconds have elapsed since the start of the application of the secondvoltage.

EXPERIMENT EXAMPLES

The present invention will now be described in more specific termsthrough experiment examples.

The above-mentioned biosensor system 100 was used in the followingexperiment examples. The sensor chip 200 shown in FIGS. 2, 3, and 5A wasused as the sensor chip. The sensor chip 200 is constituted as follows.

The capillary 40 was designed so that it was 1.2 mm wide, 4.0 mm long(deep), and 33 to 150 μm high. The height H was confirmed by slicing thesensor chip 200 and using a microscope of measure the distance from theworking electrode 11 to the ceiling of the capillary 40 (the inner faceof the cover 203).

Polyethylene terephthalate was used for the insulated board 201. Theelectrodes 11 to 13 were each formed by vapor depositing palladium onthe insulated board 201, and making slits with a laser so that thesurface area of the working electrode 11 inside the capillary 40 was 1.0mm², and the surface area of the counter electrode 12 inside thecapillary 40 was 1.2 mm².

The reagent layer 20 was formed as follows. Glucose dehydrogenase,potassium ferricyanide (made by Kanto Chemical), and taurine (made byNakalai Tesque) were used. An aqueous solution was prepared bydissolving glucose dehydrogenase so that the glucose dehydrogenaseconcentration in the reagent layer 20 would be 2.0 U/sensor chip. Thepotassium ferricyanide and the taurine were dissolved in this aqueoussolution in amounts of 1.7 wt % and 1.0 wt %, respectively, to obtain areagent solution. This reagent solution was applied over the insulatedboard 201, and the coating was dried in an atmosphere with a humidity of45% and a temperature of 21° C.

In the following experiment examples, unless otherwise specified, a timeof “0” on the graph is the point when the introduction of a sample wasdetected. Also, the temperature in the following experiment examples isthe air temperature of the measurement environment. Blood adjusted to aspecific glucose value was used as the sample to be measured.

Experiment Example 1

In this experiment example, the same processing as in FIG. 7 wasperformed with the above-mentioned biosensor system 100, except thatstep S13 was omitted. That is, steps S11, S12, and S14 to S16 in FIG. 7were performed. More specifically, after step S12, a constant voltage of250 mV was applied, and the response current value (sometimes referredto simply as “current value”) was measured. The current value for eachsensor chip was measured using sensor chips with different heights H ofthe capillary 40. More specifically, the height H of the capillary 40was either 150 μm, 100 μm, 59 μm, or 33 μm.

FIGS. 11A to 11D show the results of measuring current value using bloodwith a glucose concentration of 100 mg/dL (deciliter). FIGS. 13A to 13Dshow the results of measuring current value using blood with a glucoseconcentration of 400 mg/dL (deciliter). FIG. 12A and FIGS. 11A to 11Dare graphs of the current value at a point when 8 seconds have elapsed.FIG. 12B is a graph of the variance in the current value at varioustemperatures, when the current value at 21° C. in FIG. 12A was 0%.

As shown in FIGS. 11A and 11B and FIGS. 13A and 13B, when the height His high, the current value will be large under high temperatureconditions and will be small under low temperature conditions regardlessof the measurement time (the elapsed time after the start of thereaction).

Meanwhile, as shown in FIGS. 11C and 11D and FIGS. 13C and 13D, when theheight H is low (59 μm or 33 μm), a larger current value was measuredunder high temperature conditions than under low temperature conditionswhile the measurement time was short, but when the measurement time waslonger, the a larger current value was measured under low temperatureconditions than under high temperature conditions. The reason for thisinversion seems to be that with a finite diffusion system, under hightemperature conditions more of the substrate (glucose) is consumed in ashort period, so the substrate is used up over time, but under lowtemperature conditions there is substrate left over.

Because this inversion occurs, as shown in FIGS. 12A and 12B and FIGS.14A and 14B, in this experiment example there was a large variance inthe current value when the temperature changed even when the height Hwas 33 μm, that is, when the height H was low.

Experiment Example 2

The measurement results for current value in Experiment Example 1 wereintegrated every 0.1 second to calculate the amount of charge. Theresults are shown in FIGS. 15A to 15D and FIGS. 17A to 17D. FIGS. 15A to15D correspond to FIGS. 11A to 11D, and FIGS. 17A to 17D correspond toFIGS. 13A to 13D. FIGS. 16A and 18A are graphs of the amount of chargeafter 8 seconds have elapsed in FIGS. 15A to 15D and FIGS. 17A to 17D,respectively, and FIGS. 16B and 18B are graphs of the variance in theamount of charge due to temperature in FIGS. 16A and 18A, respectively.

As shown in these graphs, even if the height H was low, there was alarge amount of variance in the amount of charge when a constant voltagewas applied.

Experiment Example 3

The response current value was measured with the measurement voltage instep S14 set at 250 mV and the period in which the applied voltage instep S13 in FIG. 7 was an open circuit voltage (open). The conditionsother than voltage, namely, the height H of the capillary, thetemperature conditions, the glucose concentration of the sample, and soforth, were the same as the conditions in Experiment Example 1.

FIGS. 19A to 19D show the measurement results for current value whenblood with a glucose concentration of 100 mg/dL (deciliter) was used asthe sample. As shown in FIGS. 11A to 11D, the lower was the height ofthe capillary 40, the less variance there was in the measurement resultsdue to environment temperature. In particular, there was little variancewhen the height H was 59 μm or less, and variance was least at 33 μm.

FIG. 20A is a graph of the current value at a point when 8 seconds hadelapsed in FIGS. 19A to 19D (a point when 3 seconds had elapsed afterthe start of the voltage application of 250 mV). FIG. 20B is a graph ofthe variance in the current value at various temperatures when thecurrent value at 21° C. in FIG. 20A was used as a reference value (0%).

As shown in FIGS. 20A and 20B, when the height H is 59 μm, the variancedue to temperature was kept to less than ±20%. When the height H was 33μm, the variance was kept to let than ±10%.

The same experiment was conducted using a sample in which the glucoseconcentration was 400 mg/dL. As shown in FIGS. 21A to 21D and FIGS. 22Ato 22B, even when the glucose concentration in the sample was high, thevariance was kept small when the height H was 59 μm or less, andparticularly when it was 33 μm.

As discussed above, if the height H of the capillary is merely reduced,inversion will occur between the current value at high temperature andthe current value at low temperature as the measurement time passes, andvariance in the current value due to differences in temperature will notbe suppressed.

In contrast, in this experiment example, the mediator is maintained in astate in which no electrons are transmitted to the working electrode 11inside the capillary 40, by having the applied voltage be open circuitvoltage for 5 seconds after the start of the enzyme reaction. Since theenzyme reaction proceeds during this time as well, electrons areaccumulated in the mediator. After this, the measurement voltage V_(mes)is applied and the concentration measured at the point when electronswere accumulated. As a result, the current value during concentrationmeasurement is the sum of adding the amount of reaction during theapplication of the open circuit voltage to the amount of reaction duringthe application of the measurement voltage V_(mes). As a result, it isbelieved that fluctuation in the measured value caused by theenvironment temperature during measurement was kept small.

Experiment Example 4

In this experiment example, it was confirmed that the same effect as inExperiment Example 3 can be obtained even when the open circuit voltageis applied for a different length of time. In this experiment example,the duration of application of the open circuit voltage was 1.5 secondsor 3 seconds. Also, the glucose concentration was 40 mg/dL, 155 mg/dL,345 mg/dL, or 600 mg/dL. In FIGS. 23A to 23D, FIGS. 24A to 24D, FIGS.25A to 25D, and FIGS. 26A to 26D, the duration of application of theopen circuit voltage is 1.5 seconds. In FIGS. 28A to 28D, FIGS. 29A to29D, FIGS. 30A to 30D, and FIGS. 31A to 31D, the duration of applicationof the open circuit voltage is 3 seconds. In all of these drawings, theoperation that was carried out is the same except that the glucoseconcentration is different.

As shown in FIGS. 23A to 23D, FIGS. 24A to 24D, FIGS. 25A to 25D, FIGS.26A to 26D, FIGS. 28A to 28D, FIGS. 29A to 29D, FIGS. 30A to 30D, andFIGS. 31A to 31D, even when there is a change in the glucoseconcentration and/or the open circuit voltage application duration,variance in the current value due to temperature was small when theheight H was 59 μm or less, and particularly when it was 33 μm.

Also, as shown in FIGS. 27A to 27D, when the height H was 59 μm or less,the current value was not affected greatly by temperature. That is,accurate measurement results were obtained, with little error due totemperature. In particular, variance was extremely small when the heightwas 33 μm. If variance is small, then even if the temperature isdifferent, the concentration can be calculated from a single calibrationcurve. As shown in FIGS. 32A to 32D, the same results were observed forthe current value at the point when 7 seconds had elapsed in FIG. 29(the measurement results when the open circuit voltage applicationduration was 3 seconds and the glucose concentration was 155 mg/dL).

Experiment Example 5

In the following Experiment Examples 5 to 9, the glucose concentrationis 100 mg/dL.

In Experiment Example 5, the response current value was measured whenthe height H of the sensor chip is 150, 104, 90, 82, 69, 59, 49, or 33μm, the duration of application of the open circuit voltage was 0, 1,1.5, 2, 3, 4, 5, 6, or 7 seconds, and the 250 mV measurement voltage wasapplied after the application of the open circuit voltage.

When FIGS. 33A to 34D, which show the results when no open circuitvoltage was applied (when the application duration was 0 seconds), arecompared to FIGS. 35A to 36D, which show the results when an opencircuit voltage was applied, it can be seen that variance in the currentvalue due to temperature was kept smaller when the open circuit voltagewas applied. This effect is more pronounced when the open circuitvoltage was applied for at least 1.5 seconds, and even more so when theapplication was for at least 2 seconds.

Data is shown in the drawings for when the open circuit voltage wasapplied for up to 5 seconds, but variance in the current value due totemperature was similarly kept small when the application length was 6or 7 seconds.

Experiment Example 6

The height H of the sensor chip was 150, 104, 90, 82, 69, 59, 49, or 33μm. The duration of application of the open circuit voltage was 0 or 2seconds. The response current value was measured for when the 250 mVmeasurement voltage was applied after the application of the opencircuit voltage.

Under the various conditions, the current value was measured at 5° C.,14° C., 21° C., 30° C., and 38° C., and the discrepancy (variance) inthe result obtained at each temperature from the measurement result at21° C. was calculated. In FIGS. 47A to 50D, the discrepancy when theheight H is 150 μm is expressed by a broken line, and the discrepancywhen the height H is 104, 90, 82, 69, 59, 49, or 33 μm is expressed by asolid line.

That is, the solid-line curve at the top in FIG. 49A shows how much thecurrent value obtained when a measurement voltage of 250 mV was appliedafter the application of an open circuit voltage for 2 seconds (openduration) at 38° C., with a sensor chip having a height H of 104 μm,deviates from the current value measured at 21° C. (measured under thesame conditions except for the temperature). That is, the closer thesolid line is to the 0% horizontal axis, the smaller is the variance.The broken line at the top in this same drawing shows how much thecurrent value measured at a height H of 150 μm and 38° C. deviates fromthe current value measured at 21° C.

As shown in FIGS. 47A to 50C, when the open circuit voltage applicationduration was 2 seconds, the variance due to temperature was kept smallerthan when the duration was 0 seconds.

As shown in FIGS. 49A to 49D and FIGS. 50A to 50C, the variance incurrent value when the height H is 104 μm is quite different from thevariance in current value when the height H is 150 μm (FIG. 49A). Incontrast, when the height H is 90 μm or less, the variance in currentvalue due to temperature was kept smaller than when the height H was 104μm (FIG. 49B, etc.). Specifically, the height H of the capillary ispreferably 90 μm or less.

Although not shown in the data graphs, the variance was similarly keptsmall when the open circuit voltage application duration was 3 to 5seconds.

Experiment Example 7

In this experiment example, a measurement voltage of 250 mV was appliedafter a voltage of 100 mV had been applied for 3 seconds to sensor chipsof different heights H. The heights H were 150, 104, 90, 82, 69, 59, 49,and 33 μm.

As shown in FIGS. 51A to 51D and FIGS. 52A to 52D, this experimentexample makes it clear that there is still a variance suppression effecteven when a voltage that is lower than the measurement voltage isapplied instead of an open circuit voltage. The variance suppressioneffect was particularly pronounced when the height H was 59 μm or less.

Experiment Example 8

In this experiment example, a measurement voltage of 250 mV was appliedafter a voltage of 0 mV had been applied for 3 seconds to sensor chipsof different heights H. The heights H were the same as in ExperimentExample 7.

As shown in FIGS. 53A to 53D and FIGS. 54A to 54D, there is still avariance suppression effect even when a voltage of 0 mV is appliedinstead of an open circuit voltage. The variance suppression effect wasparticularly pronounced when the height H was 59 μm or less.

Experiment Example 9

In this experiment example, a measurement voltage of 250 mV was appliedafter a voltage of 250 mV had first been applied for 1 second to sensorchips of different heights H, followed by the application of an opencircuit voltage for 2 seconds. The heights H were the same as inExperiment Example 7.

As shown in FIGS. 55A to 55D and FIGS. 56A to 56D, there is still avariance suppression effect even when a closed circuit voltage isapplied prior to the application of an open circuit voltage. Thevariance suppression effect was particularly pronounced when the heightH was 59 μm or less.

Furthermore, it was confirmed that if an open circuit voltage is appliedand/or a voltage lower than the measurement voltage is applied after theapplication of a voltage higher than the measurement voltage, there wasless variance due to temperature in the current value obtained whenmeasurement voltage was applied subsequently to this.

INDUSTRIAL APPLICABILITY

The sensor chip, the biosensor system comprising this sensor chip, themethod for measuring the temperature of a biological sample, and theconcentration measurement method of the present invention all have theeffect of allowing concentration measurement error attributable totemperature to be effectively suppressed, and therefore can be widelyapplied in various fields that require more accurate measurement.

REFERENCE SIGNS LIST

-   -   11 working electrode (electrode)    -   12 counter electrode (electrode)    -   13 detection electrode    -   16 air vent    -   17 introduction port    -   20 reagent layer    -   40 capillary    -   100 biosensor system    -   101 measurement device    -   102 mounting opening    -   103 display section    -   200 sensor chip    -   201 insulated board    -   202 spacer    -   203 cover    -   204 cut-out    -   300 control circuit    -   301 a, 301 b, 301 c connector    -   302 switching circuit    -   303 current/voltage conversion circuit    -   304 analog/digital (A/D) conversion circuit    -   305 reference voltage source (first voltage applicator, second        voltage applicator)    -   306 computer (concentration measurement section, first voltage        applicator, second voltage applicator)

1-10. (canceled)
 11. A method for measuring the concentration of ananalyte in a liquid sample using a redox enzyme or an electron-transfermediator, which is executed by a biosensor system having a sensor chipcomprising a capillary into which a liquid sample is introduced, whoseheight is less than the maximum value of the sum of the diffusiondistance of the electron-transfer mediator and the diffusion distance ofthe analyte at the upper limit of the measurement guaranteed temperatureof the biosensor system, a plurality of electrodes disposed within thecapillary, and a reagent layer that is disposed within the capillary andincludes the electron-transfer mediator, said measurement methodcomprising: a first voltage application step of applying a first voltageto the electrodes; a current detection step of detecting the value ofcurrent flowing through the liquid sample during the application of thefirst voltage; a concentration measurement step of measuring theconcentration of the analyte on the basis of the current value; and asecond voltage application step of applying a second voltage to theelectrodes prior to the detection of the current value, so that thetemperature of the liquid sample will have less effect on themeasurement results of the concentration measurement section.
 12. Themeasurement method according to claim 11, wherein the second voltage isset such that electrons will be accumulated in the electron-transfermediator by the application of the second voltage.
 13. The measurementmethod according to claim 11, wherein the second voltage is an opencircuit voltage.
 14. The measurement method according to claim 11,wherein the first voltage is a voltage of positive polarity, and thesecond voltage is a voltage that is lower than the first voltage. 15.The measurement method according to claim 11, wherein the concentrationmeasurement step includes a calculation step of using a calibrationcurve or table that correlates the current value and the analyteconcentration, and calculating the analyte concentration on the basis ofthe same calibration curve or table even if the temperature of theliquid sample should fluctuate.
 16. The measurement method according toclaim 11, wherein the height of the capillary of the sensor chip is nomore than 90 μm, and in the concentration measurement step, theconcentration of the analyte is measured on the basis of the currentvalue at a point when no more than 10 seconds have elapsed since thestart of the application of the second voltage.